Unveiling the molecular mechanisms of the type IX secretion system's response regulator: Structural and functional insights

Abstract The type IX secretion system (T9SS) is a nanomachinery utilized by bacterial pathogens to facilitate infection. The system is regulated by a signaling cascade serving as its activation switch. A pivotal member in this cascade, the response regulator protein PorX, represents a promising drug target to prevent the secretion of virulence factors. Here, we provide a comprehensive characterization of PorX both in vitro and in vivo. First, our structural studies revealed PorX harbors a unique enzymatic effector domain, which, surprisingly, shares structural similarities with the alkaline phosphatase superfamily, involved in nucleotide and lipid signaling pathways. Importantly, such pathways have not been associated with the T9SS until now. Enzymatic characterization of PorX's effector domain revealed a zinc-dependent phosphodiesterase activity, with active site dimensions suitable to accommodate a large substrate. Unlike typical response regulators that dimerize via their receiver domain upon phosphorylation, we found that zinc can also induce conformational changes and promote PorX's dimerization via an unexpected interface. These findings suggest that PorX can serve as a cellular zinc sensor, broadening our understanding of its regulatory mechanisms. Despite the strict conservation of PorX in T9SS-utilizing bacteria, we demonstrate that PorX is essential for virulence factors secretion in Porphyromonas gingivalis and affects metabolic enzymes secretion in the nonpathogenic Flavobacterium johnsoniae, but not for the secretion of gliding adhesins. Overall, this study advances our structural and functional understanding of PorX, highlighting its potential as a druggable target for intervention strategies aimed at disrupting the T9SS and mitigating virulence in pathogenic species.

This study ΔporN in strain W83 (6) Plasmids pET28a (Kan R ) Novagen pRK2013 (IncP Tra + Km r ) (3) pYT313 (Ap r (Em) r ) (7) pYT377 (1.9 kb region upstream of gldK cloned in pYT313) This study pYT379 (3 kb region downstream of gldO cloned in pYT377) This study pIM03 (2 kb region upstream of porXFJ cloned in pYT313) This study pIM06 (1.9 kb region downstream of porXFJ cloned in pIM03) This study pCP23 (Ap r (Tc) r ) (8) pIM10 (2.0 kb region spanning porXFJ cloned in pCP23) This study pUC19 (Amp R ) NEB pT-COW (Amp R and Tc R in E. coli; Tc R in P. gingivalis; pVA2198 (Em r and Sp r ) (2) pTCOW-groES-porXPG     The alignment was prepared using Clustal Omega (9) and ESPript (10).The receiver, three-helix bundle and alkaline phosphatase domains are labelled in cyan, yellow and green respectively.Reference amino acid numbering and secondary structure prediction is according to the PorXFJ sequence.The secondary structure alignment in blue and light green correspond to the alternate chain conformations of the primitive orthorhombic dimeric structure with ⍺* indicating the area of conformational difference between the two chains.Amino acid residues marked with a star correspond to functionally significant residues whose mutations are discussed in this manuscript.Alternatively, AcP was found to induce cyclization of Asp54 and Lys104, resulting in a dehydration reaction ("-1 H20" labels).A rabbit polyclonal anti-PorXPG primary antibody and a polyclonal goat anti-rabbit horseradish peroxidase-conjugated secondary antibody were employed to detect PorX (~61 kDa).The biotinylated protein MmdC (~15 kDa) was used as a loading control (6) and was detected using horseradish peroxidase conjugated Streptavidin.
The anti-PorXPG did not recognize PorXFJ due to sequence variations that affect epitope recognition.Among the truncation variants, PorXPG-REC (~14 kDa) and PorXPG-REC+THB (~24 kDa) were not detected, while the PorXPG-THB+APS variant (~46 kDa) was identified by the anti-PorXPG.This selective recognition of the APS domain truncation variant by the anti-PorXPG antibody may be attributed to the presence of recognizable epitopes exclusively within the APS domain.Nonetheless, the possibility of low expression levels or the instability and subsequent degradation of the PorXPG-REC and PorXPG-REC+THB truncation variants cannot be excluded.

Isothermal titration calorimetry (ITC)
Binding affinity measurements were performed at 20°C using an isothermal titration calorimeter (Microcal iTC200, Malvern).50 M PorXFJ was diluted in buffer E and placed in the sample cell.Buffer E supplemented with 750 M zinc chloride was placed in the syringe and titrated into the protein samples.Each titration was 10 l in volume and lasted for 10.3 sec followed by an equilibration period of 240 s.

Inductively coupled plasma mass spectrometry (ICP-MS)
PorXFJ variants and method blanks, were digested in PTFE vessels using trace metal grade concentrated nitric acid at room temperature overnight, followed by 2 h at 90°C.Digestates were diluted with deionized water to 2 % w/w nitric acid and the concentrations of Mg, Mn, Co, Zn, and Cd were determined by ICP-MS.A Thermo X-Series 2 ICP-MS with collision cell technology (CCT) and chilled spray chamber was used in kinetic energy discrimination (KED) mode with 8 % hydrogen in helium to reduce interferences.The protein-divalent metal cation stoichiometries were determined by dividing the total number of the protein and each divalent metal cation number of moles.

Phosphodiesterase activity assay in vitro
PorXFJ variants (2.5 M wild-type or mutants) in buffer G (50 mM Tris pH 8, 150 mM sodium chloride) were screened for their phosphodiesterase activity.Similarly, the phosphorylated variants were incubated in buffer G supplemented with 20 mM AcP and 5 mM MgCl2 for 1 h at 37°C prior to measurements.For metal screening, wild-type PorXFJ was incubated in buffer G alone or supplemented with 0.5 mM ZnCl2, CuCl2, MnCl2, MgCl2, or CaCl2.For activity pH screening, phosphorylated and non-phosphorylated PorXFJ were incubated in 150 mM NaCl, 3 μM ZnCl2, supplemented with 50 mM acetic acid (pH 6), 50 mM Tris (pH 7-9) or CAPS buffer (10)(11) for 30 min at 37°C.The catalysis of bis(4-nitrophenyl) phosphate (bis-pNPP), p-nitrophenyl phosphate (pNPP) or p-nitrophenyl sulphate (pNPS) (0-10 mM) and the formation of pnitrophenol product were monitored at 405 nm on a Synergy H1 microplate reader (BioTek).The bis-pNPP reaction was monitored for 2 h at 37°C, while the pNPP and pNPS reactions were monitored for 3 days at 37°C.All assays were performed in quadruplicates.However, the absorbance measurements of the T271V mutant could not be obtained for bis-pNPP concentrations exceeding 5 mM.

Protein phosphorylation assay in vitro
For intact protein mass spectrometry, purified PorXFJ variants were incubated at 100 M in buffer F (25 mM Tris pH 7.5, 10 mM MgCl2, 1 mM MnCl2, 2 mM dithiothreitol, 5 mM AcP, 0.01 mM sodium orthovanadate).After 20 min at 37°C (or 1h at room temperature for PorXFJ D54A/T271V due to precipitation issues), the reactions were quenched by flash-freezing in liquid nitrogen.

Protein dimerization assay in vitro
PorXFJ variants (100 M wildtype or mutants) were incubated in buffer E supplemented with either 300 M ZnCl2 or 20 mM acetyl phosphate (AcP) and 5 mM MgCl2 for 1 h at room temperature.The mixture was then subjected to size exclusion chromatography (Superdex 200 increase 10/300 GL or Superdex 75 increase 10/300 GL), pre-equilibrated with buffer E.

Functional characterization in F. johnsoniae
Bacterial strains, plasmids, and growth conditions F. johnsoniae UW101 (4, 5) was the wild-type strain used in this study.F. johnsoniae strains were grown at 25° in CYE liquid (11) or TYES (12).For solid media, 15 g agar was added per liter.E. coli strains were grown in Luria-Bertani medium (LB) at 37°C (13).For most experiments, F. johnsoniae strains were propagated from −80 o C glycerol stocks on CYE agar and incubated for 48-72 h at 25°C before they were used as starter cultures.All primers and plasmids used in this study are listed in Table S1 and S2, respectively.Antibiotics were used at the following concentrations when needed: ampicillin, 100 μg/ml; erythromycin, 100 μg/ml; kanamycin, 35 μg/ml; and tetracycline, 20 μg/ml unless indicated otherwise.

Construction of the deletion mutants in F. johnsoniae
For deletion of porXFJ (Fjoh_2906), a 2.0-kbp fragment spanning part of Fjoh_2905 and the first 105 bp of porXFJ was amplified using primers 0076 (introducing a BamHI site) and 0077 (introducing an XbaI site), and the Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA).The fragment was digested with BamHI and XbaI and ligated into pYT313 (14), which had been digested with the same enzymes, to generate pIM03.A 1.9-kbp fragment spanning Fjoh_2907, Fjoh_2908, and the last 45 bp of porXFJ was amplified using primers 0167 (introducing an XbaI site) and 0079 (introducing a SalI site).The fragment was cloned into XbaI and SalI sites of pIM03 to generate the deletion construct pIM06.Plasmid pIM06 was introduced into F. johnsoniae UW101 by triparental conjugation as previously described ( 15).An erythromycinresistant clone was streaked for isolation, and grown overnight in CYE liquid with shaking at 25°C in the absence of antibiotics.These cells were plated on CYE agar containing 5% sucrose and incubated at 25°C for 2-3 days.Sucrose-resistant colonies were streaked for isolation and screened by PCR using primers 0086 and 0087, which flank porXFJ, to identify the deletion mutant CJ4057.
The same procedure was used to delete gldKLMNO using the plasmids and primers listed in Table S1 and Table S2, respectively.

Complementation of the porX deletion mutant
Primers 0086 (introducing a BamHI site) and 0087 (introducing an XbaI site) were used to amplify a 2009-bp fragment spanning porX with its putative promoter.The fragment was digested with BamHI and XbaI and ligated into pCP23 (8), which had been digested with the same enzymes, to generate pIM10.The plasmid was transferred to the porX mutant by triparental conjugation.
Tetracycline was used for screening of the complemented colonies.

Analysis of cell motility
Cells of the wild type F. johnsoniae and porXFJ deletion mutant were grown for 17 h at 25 o C in motility medium (16) without shaking.Tunnel slides were constructed using double stick tape, glass microscope slides, and glass coverslips, as previously described (17).Ten microliters of cultures were introduced into the tunnel slides, incubated for 5 min, and observed for motility using an Olympus CX41 phase-contrast microscope at room temperature (22 o C).Images were recorded using a Moment CMOS camera and analyzed using Ocular (Teledyne Photometrics, Tuscon, AZ).Rainbow traces of cell movements were made using Fiji (https://imagej.net/)and the macro-Color FootPrint (18).

Growth of F. johnsoniae on chitin
Chitin powder (practical grade from shrimp shells; Sigma C7170) was prepared as a 1% slurry essentially as described previously (19) and used as a stock solution to prepare the chitin media.Cells of F. johnsoniae were streaked on fresh TYES agar and incubated at 25°C for 2 d.
Cells were scraped off the plates, suspended in 1 ml Stanier medium (20), pelleted by centrifugation at 4,200 × g for 3 min to remove the residual TYES medium, and resuspended in Stanier medium to a concentration (OD600) of 1.0.Then 0.1 ml of the inoculation cell suspension was introduced into 50 ml of Stanier medium supplemented with 0.05% (w/v) chitin in 250-ml flasks, and incubated at 25°C with shaking (200 rpm).2.5 μg/ml of tetracycline was added for growth of the complemented strain.At various times, 1 ml samples were removed.Cells and residual chitin were collected by centrifugation at 17,000 × g for 10 min.Growth was assessed by determining the total cellular protein in the pellets using the Bradford assay as previously described (21).

Fig. S1 -
Fig. S1 -PorXFJ structure in two crystal forms.Asymmetric unit composition of (A) primitive orthorhombic and (B) primitive monoclinic crystal forms.(C) Superposition of the three dimers shown in panels A and B. Overlay of the six monomers shown in A and B aligned according to their (D) alkaline phosphatase superfamily (APS) domain and (E) receiver (REC) domain.

Fig. S3 -
Fig. S3 -The crystal structure of PorXFJ adopts the active phosphorylated-like conformation.(A) Superposition of the primitive orthorhombic crystal form of PorXFJ with the active (PDB ID:1FQW) and inactive (PDB ID: 2CHE) conformations of the CheY response regulator reveal an active-like conformation of PorXFJ, as per the positioning of the conserved Thr-Tyr pair, marked by arrows.Inactive and active conformations of CheY are colored in tan and pink, while chains A and B of PorXFJ dimer are colored in blue and grey, respectively.Specific residue labels are according to PorXFJ sequence.(B) Electron density of phosphate analogs, BeF3 and SO4 2-, at the REC domain phosphorylation site in the different crystal forms of PorXFJ.Orange, green and red spheres represent Ca 2+ , Mg 2+ and H2O respectively.

Fig. S4 -
Fig.S4-Multiple sequence alignment of PorXFJ and PorXPG.The alignment was prepared using Clustal Omega(9) and ESPript(10).The receiver, three-helix bundle and alkaline phosphatase domains are labelled in cyan, yellow and green respectively.Reference amino acid numbering and secondary structure prediction is according to the PorXFJ sequence.The secondary structure alignment in blue and light green correspond to the alternate chain conformations of the primitive orthorhombic dimeric structure with ⍺* indicating the area of conformational difference between the two chains.Amino acid residues marked with a star correspond to functionally significant residues whose mutations are discussed in this manuscript.

Fig. S6 -
Fig. S6 -The structures of PorXFJ and PorXPG (PDB code:7PVK) exhibit a highly conserved fold.(A) A superimposition of a representative monomer and a representative dimeric assembly.(B) Superimposition of the active site of the APS domain.Ligand analogs BeF3 and sulphate ions observed in the PorXFJ structures directly overlap or are situated in close proximity to the phosphoguanylyl-(3′→5′)-guanosine (pGpG) bound to PorXPG.

Fig. S7 -
Fig. S7 -Monophosphatase and sulphatase activities of PorXFJ.Catalytic activities against pnitrophenyl phosphate (p-NPP) and p-nitrophenyl sulphate (p-NPS) substates were recorded in the presence of zinc after three days.

Fig. S9 -
Fig. S9 -Proposed phosphorylation mechanism by acetyl phosphate in vitro.Reaction of active D54 with AcP can lead to (A) phosphorylation and/or (B) cyclization involving the formation of an internal peptide linkage between D54 and K104.

Fig. S10 -
Fig. S10 -Expression levels of PorX in P. gingivalis.(A) point mutations and (B) truncation variants.Whole cell lysates underwent SDS-PAGE separation followed by Western blot analysis.A rabbit polyclonal anti-PorXPG primary antibody and a polyclonal goat anti-rabbit horseradish peroxidase-conjugated secondary antibody were employed to detect PorX (~61 kDa).The biotinylated protein MmdC (~15 kDa) was used as a loading control (6) and was detected using horseradish peroxidase conjugated Streptavidin.

Table S1 -
Primers used in this study.

Table S3 -
Crystallization conditions and data collection parameters

Table S4 -
Data refinement statistics of the crystals.Statistic values present in parentheses correspond to the highest resolution shell data.

Table S5 -
Intact protein LC-MS analyses of PorXFJ variants in the absence or presence of phosphorylation in vitro.Expected molecular weights for protein variants (theoretical averages) vs. observed peaks before or after AcP/Mg 2+ reaction (+ 79.9 single phosphorylation; + 159.8 dual phosphorylation; -18.0 dehydration; + 61.9 combined dehydration with single phosphorylation).
*Note: Peak also observed at 60545.4 for L113E variant was likely due to pre-existing phosphorylation at T271 prior to incubation with acetyl phosphate (AcP).